The broad aims of this research are to develop new protease inhibitors which can be used to investigate the molecular mechanisms underlying the pathways of intracellular protein degradation in skeletal muscle. Initially we have synthesized eight tripeptide analogs of pepstatin with the general structure Cbz-valylvalylstatine. These compounds have been tested for their ability to inhibit pepsin and cathepsin D, to inhibit intracellular protein degradation in cultured cells, and to inhibit pH-dependent autolysis in cell homogenates. The results show that the absolute configuration and the side-chain of the unusual amino acid (statine) are important to the inhibitory action of pepstatin. The objectives of this proposal are to synthesize peptides that are analogs and derivatives of the naturally occurring protease inhibitors pepstatin and leupeptin. For the pepstatin analogs we will increase the hydrophobicity of the unusual amino acid by synthesizing the phenylalanine and tyrosine derivatives. We also plan to investigate the role of secondary binding sites by altering the absolute configuration and sequence of the N-terminal Valylvaline dipeptide. Leupeptin has the sequence leucylleucylargininal. We plan to synthesize analogs in which LeuLeu is replaced by LeuPhe, LeuTyr, LeuAla, and LeuVal. We intend to refine our testing procedures by characterizing a muscle cell culture system.